KI Platygyra Manuscript

1 Summary

Coral reefs, which already live on the edge of their thermal tolerance (1), are under acute threat from ocean warming (2, 3, 4). Corals live in symbiosis with an extraordinarily diverse genus of photosynthetic dinoflagellates (Symbiodinium spp.; 5, 6). The symbiotic association and diversity of various taxa of Symbiodinium can be flexible over time (7, 8, but see 9, 10), and individual Symbidoinium taxa can range from parasites to mutualists in their interaction with their coral host (11). Warming causes the breakdown of coral symbiosis, causing coral “bleaching” when symbionts are expelled and the white coral skeleton is visible through the coral tissue (12). Coral bleaching can lead to mortality, although corals can regain their symbionts after heat stress has abated (13, 14). The 2015/16 El Nino is the worst pulse warming event on record in terms of severity and longevity (15, 16), yet despite massive coral mortality, some corals show resilience to this extreme event (???). Here, we track coral symbioses and survival at the epicenter of this bleaching event (Kiritimati, Central Pacific), and show, contrary to our current paradigm of coral bleaching and recovery dynamics, that some corals have the capacity to re-establish symbiosis before heat stress subsides. Furthermore, we demonstrate potential mechanisms for coral survival and recovery, including the lack of preferential symbiont expulsion, and the effect of local human disturbance on pre-bleaching symbiont community structure and the probability of coral survival. Together, these results show the potential for reef corals to survive extreme warming events, providing tentative hope for the survival of corals in the Anthropocene.

2 Main Text

The symbiosis between coral and their single-celled dinoflagellate symbionts, Symbiodinium, is the foundation of reef ecosystems, and a critical element of reef resilience (van oppen and gates 2006). Corals host a diverse community of Symbiodinium, ranging along a continuum from ‘selfish opportunistic symbionts’ (e.g. some clade D Symbiodinium) which are better suited to sustained environmental stress, to ‘intimately evolved symbionts’ which provide exceptional amounts of nutrition to their coral host (17 , 18). Diversity with the genus Symbiodinium is high, described as “on the order of orders” (CITE), and is divided hierarchically into clades, subclades, and types. Symbiotic flexibility and stability can be variable both within and among coral species (Putnam et al 2012, CITE?), and this can be a driver in determining “winers and losers” (Loya et al 2001) during coral bleaching events. Coral bleaching is the breakdown of symbiosis, where Symbiodinium are expelled en masse from the tissues of their coral host. A coral’s susceptibility and resilience to bleaching is, in part, determined by fine-scale variability in their compliment of associated symbionts (Sampayo et al 2008). Corals have been observed to recover from bleaching only if the underlying stress, such as ocean warming, abates.

Ocean warming events can cause massive losses of coral cover (e.g. 17% of global coral reefs during the 1997/98 El Niño) (CITE,CITE). The 2015-2016 El Niño, superimposed on nearly-ubiquitous tropical ocean warming, instigated the third global coral bleaching event (15). Our study location, Kiritimati Atoll (Christmas Island, Kiribati, Central Equatorial Pacific, Coordinates: 2, -157.4), was at the epicenter of this extreme El Niño event. Thermal anomalies were severe on Kiritimati, rapidly exceeding NOAA Coral Reef Watch’s Coral Bleaching Alert Level 1 (4 Degree Heating Weeks, DHW, a metric of cumulative thermal stress) and Alert Level 2 (8 DHW) thresholds, reaching an unprecedented (Hoegh-Guldberg 2011) 25.7 DHW over a year-long bleaching event, demolishing most of the reef (???). Despite the massive mortality resulting from this extreme heat stress event, some corals survived.

Here, we assess coral symbiosis and survival during the massive 2015/2016 El Niño event. We tagged, sampled, and photographed the same coral colonies before, during, and immediately after the El Niño event. We assessed bleaching condition and survival for each coral colony, and used Illumina MiSeq ITS2 amplicon sequencing and 97% de novo OTU clustering to evaluate changes in Symbiodinium community structure. To investigate mechanisms underlying the ability of these corals to not only survive a year of continuous heat stress, but to recover in the interim, we assessed the relationship between human disturbance, pre-bleaching Symbiodinium community structure, and coral survival, as well as the timing of Symbiodinium community shifts throughout this El Niño event. We document, for the first time, corals that were able to visually recover from bleaching, and to regain their Symbiodinium communities during an extreme heat stress event. These corals (family Faviidae; Platygyra sp. and Favites sp.) were bleached within two months of the onset of warming, but had visibly recovered after 10 consecutive months of intense warming. Previously, corals have been shown to recover from bleaching only after the external stress (e.g. warming) has subsided (Gates and van Oppen 2016?7?), implying that longer and more frequent stressors spell disaster for reefs worldwide. This unprecedented resilience mechanism…

Figure 1

Symbiodinium clade predicts survival, and local human disturbance predisposes coral to have “suboptimal” clades. Opposed to other study which showed that only those with D survived, C didn’t, and that those which started with undetectable D and switched to C still died. Bay et al 2016 (threshold densities study) For Platygyra, Corals with clade C survived, and corals with clade D died. Symbiodinium community structure was stable within coral colonies before the onset of bleaching. The pre-bleaching distribution of Symbiodinium clade was associated with local human disturbance level, with corals at high-impact sites housing clade D as the dominant symbiont, and those at low-impact sites housing clade C as the dominant symbiont.

We do know that corals house background symbionts in low abundances (19, all the recent ngs studies…), but these relationships have been described as unstable (20, more cites?). Silverstein et al 2012 Specificity is rarely absolute Despite the consensus that coral symbioses are integral to coral resistance to and resilience from bleaching, it is thought that most background Symbiodinium types have minimal functional significance for corals (Lee et al 2016), and . . We demonstrate that tiny abundances are important - many corals who switched from C to D during the event had extremely low or undetectable sequence counts of clade D Symbiodinium before the bleaching event, but their recovery symbiont communities were dominated by clade D Symbiodinium

Our study also provides insight into the timing of symbiont community shifts in the field. Symbiodinium communities remained largely consistent within an individual coral colony from before-bleaching time points until many corals were mostly or entirely bleached. Significant symbiont community change only occurred after the coral was no longer visibly bleached, suggesting that in this bleaching event, corals experienced a wholesale expulsion of all symbionts (with no preferential expulsion), followed by competitive repopulation of the coral host. This adds evidence to support the theory that clade D Symbiodinium are competitively dominant during low symbiont abundance in warm conditions (cite people who have said this before). We used to think that bleaching might be good - ABH says that corals bleach in order to expel suboptimal Symbiodinium types in exchange for optimal symbionts during the new conditions (21, Baker:2001bf, 22). Switching and shuffling (23) And we do know that some symbio are “better” than others And then we said that bleaching is definitely bad But at least we do know that it allows changes to occur in the Symbiodinium community structure

Although massive bleaching events like this one will likely continue to cause catastrophic damage to coral reefs worldwide, mitigating local human disturbance can potentially help protect some coral species against a modest amount of ocean warming. Elucidating the mechanisms underlying changes in coral-symbiont interactions is essential to understanding the ability of the coral symbiome to adapt to the multiple stressors they now face.

3 Methods

The Methods section should be written as concisely as possible but should contain all elements necessary to allow interpretation and replication of the results. As a guideline, Methods sections typically do not exceed 3,000 words. Detailed descriptions of methods already published should be avoided; a reference number can be provided to save space, with any new addition or variation stated. The Methods section should be subdivided by short bold headings referring to methods used and we encourage the inclusion of specific subsections for statistics, reagents and animal models. If further references are included in this section, the numbering should continue from the end of the last reference number in the rest of the paper and the list should accompany the additional Methods at the end of the paper. The Methods section cannot contain figures or tables (essential display items should be included in the Extended Data).

3.1 Field Information

Kiritimati Atoll, Kiribati is located in the Central Equatorial Pacific (1.9N 157W), at the center of the El Niño 3.4 region (used to quantify El Niño presence and strength). During the 2015/2016 El Niño event, Kiritimati experienced 10 months of sustained temperature stress, causing a mass bleaching and mortality event (???).

Temperature loggers (Sea-Bird 56) were deployed around the island at 10-12m depth from 2011-2016 to measure in situ thermal stress.

3.2 Coral Tagging and sampling

In August/September 2014 seven taxa of coral were tagged along a 60m transect at 10-12m depth at 10 different sites around the atoll. At each site, 12 colonies of each focal species (Porites lobata, Montipora aequituberculata, and Pocillopora eyedouxi) were tagged and a total of 18 colonies of a varying assortment of Platygyra, Favities, Favia, and Hydnophora microconos were tagged. A photo of each coral with a ruler beside it was taken for colony measurments and characterists (i.e. bleaching) to be analyzed in the lab. The tissue samples were stored in a cooler on the boat and at night they were rinsed with fresh water and alliquoted into centrifuge tubes with Guanidinium, then stored at 4 (I havent looked to see how to put in a degree symbol yet) C. The tagged coral colonies were resampled once more before the El Niño (January/February 2015), twice during (May/April 2015 and July 2015), and once near the end (March 2016). Due to either storm waves breaking and moving coral or the tags dissapearing some of the corals were unable to be located and new corals were tagged to replenish numbers **I(KT) dont like this sentence - still working on how to word it*.

3.3 Pre-processing and sequencing

DNA extraction was performed as in Stat et al. (2009) with a few modifications. The DNA pellet was washed with 70% ethanol three times, and after the final wash was removed, the remaining ethanol was allowed to evaporate off before the DNA pellet was resuspended in Tris buffer (0.1M pH8). Cleanup by Amy here Library Prep sequencing exact information

ITS2 region - it’s annoying, but it’s the best we’ve got right now PCR and Cleanup - Amy’s method of PCR and cleanup Library Prep - Amy’s method of library prep, include Illumina Sequencing information (barcodes, etc)

3.4 Bioinformatics

Sequence QC - boku, then merge with illumina utils, max mismatch=3 Sequence clustering - denovo clustering using UCLUST in QIIME, then compare to reference database to assign taxonomy

3.5 Statistical Analysis

Alpha diversity of sequence reads, co-occurence?, beta diversity? Code will be avaible on git hub

4 References

1. Sampayo, E. M. et al. Coral symbioses under prolonged environmental change: Living near tolerance range limits. Sci. Rep. 6, 36271 (2016).

2. Hughes, T. P. et al. Climate change, human impacts, and the resilience of coral reefs. Science 301, 929–933 (2003).

3. Hoegh-Guldberg, O. et al. Coral reefs under rapid climate change and ocean acidification. Science 318, 1737–1742 (2007).

4. Baker, A. C., Glynn, P. W. & Riegl, B. Climate change and coral reef bleaching: An ecological assessment of long-term impacts, recovery trends and future outlook. Estuar. Coast. Shelf Sci. 80, 435–471 (2008).

5. Muscatine, L. & Porter, J. W. Reef corals: Mutualistic symbioses adapted to Nutrient-Poor environments. Bioscience 27, 454–460 (1977).

6. Rowan, R. & Powers, D. A. Ribosomal RNA sequences and the diversity of symbiotic dinoflagellates (zooxanthellae). Proc. Natl. Acad. Sci. U. S. A. 89, 3639–3643 (1992).

7. Baker, A. C. Flexibility and specificity in Coral-Algal symbiosis: Diversity, ecology, and biogeography of symbiodinium. Annu. Rev. Ecol. Evol. Syst. 34, 661–689 (2003).

8. Little, A. F., Oppen, M. J. H. van & Willis, B. L. Flexibility in algal endosymbioses shapes growth in reef corals. Science 304, 1492–1494 (2004).

9. Thornhill, D. J., LaJeunesse, T. C., Kemp, D. W., Fitt, W. K. & Schmidt, G. W. Multi-year, seasonal genotypic surveys of coral-algal symbioses reveal prevalent stability or post-bleaching reversion. Mar. Biol. 148, 711–722 (2006).

10. Thornhill, D. J., Xiang, Y., Fitt, W. K. & Santos, S. R. Reef endemism, host specificity and temporal stability in populations of symbiotic dinoflagellates from two ecologically dominant caribbean corals. PLoS One 4, e6262 (2009).

11. Lesser, M. P., Stat, M. & Gates, R. D. The endosymbiotic dinoflagellates (symbiodinium sp.) of corals are parasites and mutualists. Coral Reefs 32, 603–611 (2013).

12. Brown, B. E. Coral bleaching: Causes and consequences. Coral Reefs 16, S129–S138 (1997).

13. Douglas, A. E. Coral bleaching—-how and why? Mar. Pollut. Bull. 46, 385–392 (2003).

14. Stat, M., Loh, W. K. W., LaJeunesse, T. C., Hoegh-Guldberg, O. & Carter, D. A. Stability of coral–endosymbiont associations during and after a thermal stress event in the southern great barrier reef. Coral Reefs 28, 709–713 (2009).

15. Eakin, C. M. et al. Global Coral Bleaching 2014-2017: Status and an Appeal for Observations. Reef Encounter 31, 20–26 (2016).

16. Heron, S. F., Maynard, J. A., Hooidonk, R. van & Eakin, C. M. Warming trends and bleaching stress of the world’s coral reefs 1985-2012. Sci. Rep. 6, 38402 (2016).

17. Stat, M., Carter, D. & Hoegh-Guldberg, O. The evolutionary history of Symbiodinium and scleractinian hosts — symbiosis, diversity, and the effect of climate change. Perspectives in Plant Ecology 8, 23–43 (2006).

18. Lesser, M. P., Stat, M. & Gates, R. D. The endosymbiotic dinoflagellates (Symbiodinium sp.) of corals are parasites and mutualists. Coral Reefs 1–9 (2013).

19. Correa, A. M. S., McDonald, M. D. & Baker, A. C. Development of clade-specific Symbiodinium primers for quantitative PCR (qPCR) and their application to detecting clade D symbionts in Caribbean corals. Marine biology 156, 2403–2411 (2009).

20. Coffroth, M. A., Poland, D. M., Petrou, E. L., Brazeau, D. A. & Holmberg, J. C. Environmental symbiont acquisition may not be the solution to warming seas for reef-building corals. PLoS ONE 5, e13258 (2010).

21. Buddemeier, R. W. & Fautin, D. G. Coral bleaching as an adaptive mechanism. Bioscience 43, 320–326 (1993).

22. Buddemeier, R. W., Baker, A. C., Fautin, D. G. & Jacobs, J. R. in (Coral health; …, 2004).

23. Baker, A. C. Flexibility and specificity in coral-algal symbiosis: diversity, ecology, and biogeography of Symbiodinium. Annual Review of Ecology, Evolution, and Systematics 661–689 (2003).